Quantitative Real-Time PCR Analysis

RNA was extracted with TRIZOL-reagent (Invitrogen). Preparation of the cDNA (Superscript II, Invitrogen) and real time quantification of the mRNA concentrations with the Fast-Start Sybr Green I kit and the LightCycler II instrument (Roche) were done essentially as described [26 (link)], however using per assay a cDNA input derived from 75 ng of total RNA. Titration of relative copy numbers against external standards and normalization against the not regulated reference gene chloride intracellular channel 1 (CLIC1) were done as detailed in [48 (link)]. Sequences of oligo nucleotide primers are listed in Additional file 1.

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Günther J., Czabanska A., Bauer I., Leigh J.A., Holst O, & Seyfert H.M. (2016). Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages. Veterinary Research, 47, 13.

Publication 2016

TRIZOL-reagent Superscript II Fast-Start Sybr Green I kit LightCycler II

Assay Cdna Chloride intracellular channel 1 Fast green Gene Mrna Nucleotide sequences Oligo Primers Titration Trizol

Corresponding Organization :

Other organizations : Research Institute for Farm Animal Biology (FBN), Research Center Borstel - Leibniz Lung Center, University of Nottingham

Top 5 similar protocols

Variable analysis

independent variables

RNA extraction with TRIZOL-reagent (Invitrogen)
Preparation of the cDNA (Superscript II, Invitrogen)
Real-time quantification of the mRNA concentrations with the Fast-Start Sybr Green I kit and the LightCycler II instrument (Roche)

dependent variables

MRNA concentrations

control variables

CDNA input derived from 75 ng of total RNA
Normalization against the not regulated reference gene chloride intracellular channel 1 (CLIC1)

controls

External standards for titration of relative copy numbers

Annotations

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